GIF is a 13-kDa cytokine discovered in the course of the study on IgE synthesis. It is encoded by a single gene in the genome and its nucleotide sequence is identical is that published for macrophage migration inhibitory factor (MIF). MIF(GIF)-/- mice show enhanced susceptibility to Leishmania major. These mice are incapable of developing the experimental colitis. These findings support the notion that GIF is involved inactivating the innate immune system. GIF protein is expressed in the cytosol of hematopoietic and nonhematopoietic cells. GIF secreted from T cells is cysteinylated at C60, whereas that contained in the cytosol of the same cells is unmodified. The modification at C60 is required for the capability of this cytokine to bind to target cells, to inhibit BCR-mediated uptake of antigen, and to inhibit IL-4 secretion from CD4 cells. Binding assays demonstrated that GIF receptors are expressed on T and B cells after these cells are activated, but not on macrophage and dendritic cell lines. Chemical crosslinking experiments suggest that GIF binds to a 50-52 kDa cell surface protein. GIF-/- mice show enhanced antibody responses to T-dependent antigens. GIF-/- CD4 cells were polarized toward a Th2 phenotype as compared with wild type cells. To clarify the role of this cytokine in T-dependent and -independent humoral responses, GIF+/+ and -/- mice crossed to BALB/c and those to B6 will be immunized with various antigens and adjuvants. The role of GIF in cognate T-B interaction will be analyzed in vitro and in vivo using TCR Tg mice and BCR Tg mice on GIF+/+ and -/- backgrounds. The biochemical mechanisms by which GIF regulates BCR-mediated antigen presentation will be analyzed. TCR Tg GIF+/+ and -/- mice will be compared for the signals that regulate Th differentiation. These mice will also be used to determine the involvement of the innate immunity in the regulation of Th differentiation. The functional relevance of GIF-dependent Th development will be addressed in the mouse model of allergic airway inflammation. Finally, using C60-modified rGIF, GIF receptor will be molecularly characterized. These experiments will be useful to determine whether this cytokine directly regulates innate immunity or antigen-specific immunity and whether the form secreted from T cells or that contained in the cytosol plays an important role in its function. This project will fill a critical gap in our knowledge in cytokine biology.